CN118109480A - Early 15 gene and application thereof in regulating and controlling plant growth and development - Google Patents

Abstract

The invention discloses an early 15 gene, a coded protein thereof and application thereof in regulating and controlling plant growth and development, in particular to regulating and controlling early maturing and dwarf of corn. The present invention demonstrates that early maturing and dwarf plants, particularly maize plants, can be obtained by over-expressing the "early 15" gene in plants, particularly maize.

Description

Early 15 gene and application thereof in regulating and controlling plant growth and development

Technical Field

The invention relates to an early 15 gene, a coded protein thereof and application thereof in regulating and controlling plant growth and development, in particular to regulating and controlling early maturing and dwarf of corn.

Background

Corn occupies a serious position in the grain production in China and even worldwide. Early maturing, dwarf and density resistance are the directions of corn breeding. On one hand, the corn planting area can be enlarged, so that the corn planting area can be used as a spring sowing area in the cold area in the north and in the cold area in one season; on the other hand, the early maturing can lead the corn to be planted in the suitable sowing area in a repeated sowing way. In order to ensure that the corn rotation area of Huang-Huai-Hai wheat is two seasons in one year, the growth period of corn cannot be too long, and the genes with shortened growth period can enable varieties which are too long in growth period and are not suitable for being planted in Huang-Huai-Hai to become suitable for being planted in Huang-Huai-Hai summer. In addition, the early ripening is more suitable for mechanical operation, because the moisture content of the corn kernels is reduced by less than 25% before harvesting after the corn is ripe, and the direct harvesting of the kernels can lead to the increase of the kernel breaking rate of the corn kernels, which is not beneficial to mechanical harvesting. The corn dwarf can increase planting density, enhance lodging resistance and improve yield.

The transcription factor coded by MADS-box family gene plays a very important role in ABCDE model of plant flower development, and also participates in regulating other links in plant development process, such as growth of vegetative organ, etc. AGL12 (Tapia-Lopez et al, 2008) and AGL28 (Yoo et al, 2006) in Arabidopsis are related to flowering time of plants. OsMADS14 in rice is a regulator mainly responsible for early flowering of rice, and overexpression of OsMADS14 gene in rice can severely shorten the heading stage of rice and form an extremely early flowering phenotype (Pelucchi et al., 2002). The gene OsMADS15 regulating rice flowering downstream, the overexpression of which can lead to the elongation of transgenic rice internodes, the plant becoming shorter and flowering being advanced (Lu et al 2012). Overexpression of ZmMADS1 in maize resulted in an early flowering phenotype, and RNA interference mediated down-regulation of ZmMADS1 resulted in maize flowering delay (Alter et al, 2016). Unlike the ZmMADS1 sequence reported by Alter from maize inbred B73, the "early 15" gene encoding MADS-box transcription factor MADS1 is also derived from maize inbred Zheng 58, and there is 8 point differences in CDS sequence and 5 point differences in amino acid sequence from the ZmMADS1 gene reported in the literature. The 1-point difference in the amino acid sequence resides in the K domain, which is involved in mediating protein intermolecular interactions; the other 4 point differences are all located at the C-terminal C-domain, which mediates different MADS-box proteins to exert different effects.

At present, the application of the gene similar to maize 'early 15' has not been reported. The rice MADS-box gene (CN 00813062.0) has been reported in the existing domestic patent to have a function of regulating plant branching. MADS transcription factor families SILKY1 (CN 200880019049.4) and ZMM28 (CN 201980026679.2) in maize can increase grain yield. In addition, the ZmMADS15 can regulate and control the corn laying time and the corn scattering time under the condition of short sunshine. However, zmMADS15 did not affect the corn laying time and the meal time under long sun conditions (CN 115011628A).

Therefore, there is still a need to find genes which regulate plant growth and development, and have important application value especially for maize early maturing and dwarf direction breeding.

Summary of The Invention

The invention has important application value for early ripening of plants, especially corns, and dwarf direction breeding.

The inventor finds that the plant, especially the corn plant, can lead the flowering period and the maturity of the plant, especially the corn plant, to be advanced under the conditions of short sunlight and long sunlight by over-expressing the early 15 gene in the plant, especially the corn, and the plant height and the spike position to be reduced; by mutating the early 15 gene by a gene editing technology, plants with delayed flowering and maturity, especially maize plants, can be obtained.

Therefore, the invention aims to provide a maize early 15 gene, a coded protein thereof and application thereof in regulating plant growth and development, in particular to application of the gene in regulating maize early ripening and dwarf.

Thus, according to a first aspect, the present invention relates to the maize "early 15" gene.

The corn 'early 15' gene source of the invention is Zheng 58, and the Zea mays (B73 RefGen_v3) version is encoded as follows: GRMZM2G171365, zea mays (Zm-B73-REFERENCE-GRAMENE-4.0) version coded as Zm00001d048474, and Zea mays (Zm-B73-REFERENCE-NAM-5.0) version coded as Zm00001eb403750 (see FIG. 1).

Gene sequence information

The invention 'early 15' gene related sequence

GRMZM2G171365cDNA (Zheng 58) (SEQ ID NO: 1)

CGGCCATCACGGTGCGCCCTTTCCCTTCCTCCCCAGATCCCCGTCCCCGTTTTCCACTTTTGCCTCCGCCCCAATTCGGATAACAAACCCCTCCGCCTCGTCGCGTCTCCTCCCAGCCGAGCCGATCCGGTAGAGAGAGGGAGAGGGAGAGGGAGGGACTGAGGGAGGAGGAGCTGGGTTCCGGTCCCGGCCGCCCGGCCGGCTGCGCGATTCGATTGTAGCTCTCGTCCCCGGGCGGCGTCCAGGATGGTGCGGGGCAAGACGCAGATGAAGCGAATAGAGAACCCGACCAGCCGCCAGGTCACCTTCTCCAAGCGCCGCAACGGCCTGCTCAAGAAGGCGTTCGAGCTCTCCGTCCTCTGCGACGCCGAGGTCGCCCTCGTCGTCTTCTCCCCGCGCGGCAAGCTCTACGAATTCGCCAGCGGAAGTGCGCAGAAAACGATTGAACGTTATAGAACATACACAAAGGATAATGTCAGCAACAAGACAGTGCAGCAGGATATTGAGCGAGTAAAAGCTGATGCGGATGGCCTGTCAAAGAGACTTGAAGCACTTGAAGCTTACAAAAGGAAACTTTTGGGTGAGAGGTTGGAAGACTGCCCCATTGAAGAGCTGCACAGTTTGGAAGTCAAGCTTGAGAAGAGCCTGCATTGCATCAGGGGAAGAAAGACTGAGCTGCTGGAGGAGCAAGTCCGTAAGCTGAAGCAGAAGGAGATGAGTCTGCGCAAGAGCAACGAAGATTTGCGTGAAAAGTGCAAGAAGCAGCCGCCTGTGCCGATGGCTCCGCCGCCGCCTCGTGCGCCGGCAGTCGACACCGTGGAGGACGATCACCGGGAGCCGAAGGACGACGGAATGGACGTGGAGACGGAGCTGTACATAGGATTGCCCGGCAGAGACTACCGCTCAAGCAAAGACAAGGCTGCAGTGGCGGTCAGGTCAGGCTAGCAGCTAGCTCAGCCACGCACAGGCCCAATCAACGCAAGCTAGCTAGCTGAGAATAATCTTTTAGATCTCTGGTAGTGTGGAGATCGAGATGCAAGCCAAGCAATGTGATATCGCGTCGTGTGTTACCAAAAAAAAAAAAAAAAGTCAGTCAGGCCA

GRMZM2G171365CDS (Zheng 58) (SEQ ID NO: 3)

ATGGTGCGGGGCAAGACGCAGATGAAGCGAATAGAGAACCCGACCAGCCGCCAGGTCACCTTCTCCAAGCGCCGCAACGGCCTGCTCAAGAAGGCGTTCGAGCTCTCCGTCCTCTGCGACGCCGAGGTCGCCCTCGTCGTCTTCTCCCCGCGCGGCAAGCTCTACGAATTCGCCAGCGGAAGTGCGCAGAAAACGATTGAACGTTATAGAACATACACAAAGGATAATGTCAGCAACAAGACAGTGCAGCAGGATATTGAGCGAGTAAAAGCTGATGCGGATGGCCTGTCAAAGAGACTTGAAGCACTTGAAGCTTACAAAAGGAAACTTTTGGGTGAGAGGTTGGAAGACTGCCCCATTGAAGAGCTGCACAGTTTGGAAGTCAAGCTTGAGAAGAGCCTGCATTGCATCAGGGGAAGAAAGACTGAGCTGCTGGAGGAGCAAGTCCGTAAGCTGAAGCAGAAGGAGATGAGTCTGCGCAAGAGCAACGAAGATTTGCGTGAAAAGTGCAAGAAGCAGCCGCCTGTGCCGATGGCTCCGCCGCCGCCTCGTGCGCCGGCAGTCGACACCGTGGAGGACGATCACCGGGAGCCGAAGGACGACGGAATGGACGTGGAGACGGAGCTGTACATAGGATTGCCCGGCAGAGACTACCGCTCAAGCAAAGACAAGGCTGCAGTGGCGGTCAGGTCAGGCTAG

GRMZM2G171365 amino acid (Zheng 58) (SEQ ID NO: 2)

MVRGKTQMKRIENPTSRQVTFSKRRNGLLKKAFELSVLCDAEVALVVFSPRGKLYEFASGSAQKTIERYRTYTKDNVSNKTVQQDIERVKADADGLSKRLEALEAYKRKLLGERLEDCPIEELHSLEVKLEKSLHCIRGRKTELLEEQVRKLKQKEMSLRKSNEDLREKCKKQPPVPMAPPPPRAPAVDTVEDDHREPKDDGMDVETELYIGLPGRDYRSSKDKAAVAVRSG

1.3 Zheng 58 and B73 sequence comparison

The GRMZM2G171365 gene contained 1 long intron, 3 short introns in the B73 database; the gene has 4 transcripts (T01 is identical to T04), wherein T01 is longest and translates 232 amino acids. The CDS length obtained from Zheng 58 clone was most similar to the T01 CDS in the B73 database, and the GRMZM2G171365CDS (Zheng 58) sequence was 8-point different from the GRMZM2G 171365-T01 CDS (B73) sequence (see FIG. 2).

The number of translated amino acids of GRMZM2G171365CDS (Zheng 58) and GRMZM2G171365_T01 CDS (B73) are identical, but the amino acid sequence has 5 point differences: P119S, P180S, P181A, T190N, D G (fig. 3). The 1-point difference in the amino acid sequence resides in the K domain, which is involved in mediating protein intermolecular interactions; the other 4 point differences are all located at the C-terminal C-domain, which mediates different roles of different MADS-box proteins (see FIG. 3).

The present invention provides an isolated nucleic acid molecule, characterized in that it comprises a sequence selected from the group consisting of:

1) A nucleotide sequence shown in SEQ ID NO. 1;

2) A nucleotide sequence with at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the nucleotide sequence shown in SEQ ID NO. 1, wherein the encoded polypeptide has the function of regulating plant growth and development;

3) A nucleotide sequence which hybridizes under stringent conditions to the sequence shown in SEQ ID No. 1;

4) Expressing the nucleotide sequence of the same or functionally deleted or mutated protein by substituting and/or deleting and/or adding one or more nucleotides in the nucleotide sequence shown in SEQ ID NO. 1;

5) Different transcripts were produced from the nucleotide sequence shown in SEQ ID No. 1.

In another aspect, the invention also provides an isolated nucleic acid molecule, characterized in that it comprises a sequence selected from the group consisting of:

1) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 2;

2) A nucleotide sequence encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity to the amino acid sequence shown in SEQ ID NO. 2, wherein the encoded polypeptide has the function of regulating plant growth;

3) A nucleotide sequence encoding an amino acid sequence obtained by substitution and/or deletion and/or addition of one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 2, and a polypeptide encoded thereby has a function of regulating plant growth and development.

In another aspect, the invention provides an isolated nucleic acid molecule characterized in that it comprises a sequence selected from the group consisting of:

1) A nucleotide sequence shown in SEQ ID NO. 3;

2) A nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the nucleotide sequence shown in SEQ ID NO. 3, wherein the encoded polypeptide has the function of regulating plant growth and development;

3) A nucleotide sequence which hybridizes under stringent conditions to the sequence shown in SEQ ID No. 3;

4) Expressing the nucleotide sequence of the same or functionally deleted or mutated protein by substituting and/or deleting and/or adding one or more nucleotides in the nucleotide sequence shown in SEQ ID NO. 3;

5) Different transcripts were produced from the nucleotide sequence shown in SEQ ID NO. 3.

In another aspect, the invention provides an isolated polypeptide, characterized in that it is transcribed and/or expressed from a nucleic acid molecule as described in the above aspects.

In another aspect, the invention provides an isolated polypeptide comprising an amino acid sequence selected from the group consisting of seq id no:

1) An amino acid sequence shown in SEQ ID NO. 2;

2) An amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity to the amino acid sequence shown in SEQ ID NO. 2, said polypeptide having the function of regulating plant growth;

3) The polypeptide has the function of regulating plant growth and development by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 2.

In another aspect, the invention provides a recombinant vector comprising a nucleic acid molecule as described in the above aspects.

In another aspect, the invention provides a host cell comprising a nucleic acid molecule as described in the above aspect, or comprising a polypeptide as described in the above aspect, or comprising a recombinant vector as described in the above aspect.

In another aspect, the invention provides a transgenic plant characterized by comprising a nucleic acid molecule as described in the above aspect, or comprising a recombinant vector as described in the above aspect.

In some embodiments, the transgenic plant is a monocot or dicot, preferably a crop plant.

In some specific embodiments, the transgenic plant is selected from one or more of maize (Zea mays), sorghum (Sorghum bicolor), sorghum vulgare, soybean (Glycine max), wheat (Triticum aestivum), rice (Oryza sativa), cotton (such as gossypium barbadense (Gossypium barbadense), canola (Brassica campestris), cabbage (Brassica oleracea), canola (Brassica napus), mustard (Brassica juncea), barley (Hordeum vulgare), rye (SECALE CEREALE), oat (AVENA SATIVA), millet (such as pearl millet (Pennisetum glaucum)), tomato (Lycopersicon esculentum), sunflower (Helianthus annuus), potato (Solanum tuberosum), peanut (Arachis hypogaea), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), sugar beet (Beta vulgares), sugarcane (Saccharum spp), tobacco (Nicotiana tabacum) or arabidopsis (Arabidopsis thaliana), preferably arabidopsis thaliana and maize, more preferably maize.

In another aspect, the invention provides the use of an isolated nucleic acid molecule as described in the above aspect or an isolated polypeptide as described in the above aspect or a recombinant vector as described in the above aspect for regulating plant growth.

In another aspect, the invention provides the use of an isolated nucleic acid molecule as described in the above aspect or an isolated polypeptide as described in the above aspect or a recombinant vector as described in the above aspect for regulating early maturity and stumps in plants.

In some embodiments, the application comprises the steps of:

1) Introducing the nucleic acid molecule described in the above aspect or the recombinant vector described in the above aspect into a plant of interest to obtain an overexpressed transgenic plant; and

2) The plants are grown such that the over-expressed transgenic plants have early maturing and dwarf performance compared to control plants.

In another aspect, the invention provides the use of an isolated nucleic acid molecule as described in the above aspect or an isolated polypeptide as described in the above aspect or a recombinant vector as described in the above aspect for regulating late maturation of a plant.

In some embodiments, the application comprises the steps of:

1) Disruption of the "early 15" gene in plants or genes homologous to the "early 15" gene in other plants to obtain transgenic plants; and

2) The plants are grown such that the transgenic plants have late-maturing properties compared to control plants.

In some embodiments, the disruption is achieved by knocking out or knocking down the "early 15" gene.

In some embodiments, the disruption is achieved by a genome editing system of CRISPR/Cas, TALEN, ZFN or other gene editing system.

In another aspect, the invention provides the use of a nucleic acid molecule as described in the above aspect or a polypeptide as described in the above aspect or a recombinant vector as described in the above aspect for breeding plants with altered traits.

In some embodiments, the plant is a monocot or dicot, preferably a crop plant.

In some specific embodiments, the transgenic plant is selected from one or more of corn, sorghum, soybean, wheat, rice, cotton, canola, cabbage, canola, mustard, barley, rye, oat, millet, tomato, sunflower, potato, peanut, sweet potato, cassava, sugar beet, sugarcane, tobacco, or arabidopsis; preferably Arabidopsis thaliana and corn; more preferably corn.

In another aspect, the invention provides a method of regulating the growth and development of a plant comprising:

1) Introducing the isolated nucleic acid molecule of the above aspect, or the recombinant vector of the above aspect, into a plant of interest to obtain a transgenic plant; and

2) Cultivating the plant, wherein the transgenic plant has the properties of early maturing and dwarfing compared to a control plant.

In another aspect, the invention provides a method of regulating the growth and development of a plant comprising:

1) Disruption of the "early 15" gene in plants or genes homologous to the "early 15" gene in other plants to obtain transgenic plants; and

2) Cultivating the plant, wherein the transgenic plant has late-maturing performance compared to a control plant.

In another aspect, the present invention provides a method of producing a transgenic plant comprising the isolated nucleic acid molecule according to the above aspect introduced, or the recombinant vector according to the above aspect, or the isolated polypeptide according to the above aspect expressed recombinantly, characterized in that the method comprises the steps of:

1) Obtaining seeds of said transgenic plant;

2) Planting the seeds to obtain the transgenic plant with stable inheritable characters.

In another aspect, the present invention provides a method of producing a transgenic plant comprising a gene that disrupts the "early 15" gene in the plant or is homologous to the "early 15" gene in other plants, characterized in that the method comprises the steps of:

1) Obtaining seeds of said transgenic plant;

2) Planting the seeds to obtain the transgenic plant with stable inheritable characters.

In some specific embodiments, the transgenic plant is selected from one or more of corn, sorghum, soybean, wheat, rice, cotton, canola, cabbage, canola, mustard, barley, rye, oat, millet, tomato, sunflower, potato, peanut, sweet potato, cassava, sugar beet, sugarcane, tobacco, or arabidopsis; preferably Arabidopsis thaliana and corn; more preferably corn.

Drawings

FIG. 1 shows a summary of different versions of the "early 15" gene.

FIG. 2 shows CDS sequence differences for the "early 15" gene in Zheng 58 and B73.

FIG. 3 shows the amino acid sequence differences of the "early 15" gene in Zheng 58 and B73. MADS-box domain (3-75), K-box domain (83-169), C domain (170-231).

FIG. 4 shows pBCXUN vector maps.

FIG. 5 shows recombinant expression vector pBXCUN- "early 15".

FIG. 6 shows the relative expression of the "early 15" gene in leaves of control plants and transgenic plants of the invention.

FIG. 7 shows the structure of gene editing vector pXUE C.

FIG. 8 shows a comparison of the number of days to powder of control plants and transgenic inbred plants overexpressing the invention for each year. By Dunnett's test analysis, p.ltoreq.0.05; * P is less than or equal to 0.01.

FIG. 9 shows a comparison of the days of silking of control plants of each year and transgenic inbred plants overexpressing the invention. By Dunnett's test analysis, p.ltoreq.0.05; * P is less than or equal to 0.01.

FIG. 10 shows a comparison of maturity days for control plants and plants of the over-expressed transgenic inbred of the invention. By Dunnett's test analysis, p.ltoreq.0.05; * P is less than or equal to 0.01.

FIG. 11 shows a comparison of the number of days to powder of control plants and plants of the over-expressed transgenic hybrids of the present invention for each year. By Dunnett's test analysis, p.ltoreq.0.05; * P is less than or equal to 0.01.

FIG. 12 shows a comparison of the number of days of laying for each year of control plants and plants of the over-expressed transgenic hybrids of the invention. By Dunnett's test analysis, p.ltoreq.0.05; * P is less than or equal to 0.01.

FIG. 13 shows a comparison of maturity days for control plants and plants of the over-expressed transgenic hybrids of the present invention. By Dunnett's test analysis, p.ltoreq.0.05; * P is less than or equal to 0.01.

FIG. 14 shows a comparison photograph of milk lines of a control plant and an over-expressed transgenic hybrid spike of the invention (panel A is a control plant, panel B is a transgenic hybrid plant of the invention).

FIG. 15 shows photographs of flowering phase versus plant height of control plants and transgenic inbred plants of the invention (panels A and B, left control plants, right transgenic inbred plants of the invention).

FIG. 16 shows a photograph of the flowering phase versus plant height of control plants and of the over-expressed transgenic hybrid plants of the invention (panels A and B, left control plants, right transgenic hybrid plants of the invention).

FIG. 17 shows a comparison of the days to powder dispersion for control plants and gene-edited plants of the invention. By Dunnett's test analysis, p.ltoreq.0.05; * P is less than or equal to 0.01.

FIG. 18 shows a comparison of the number of days of laying for control plants and gene-edited plants of the invention. By Dunnett's test analysis, p.ltoreq.0.05; * P is less than or equal to 0.01.

Figure 19 shows a comparison of plant heights for control plants and over-expressed transgenic inbred plants of the invention for each year. By Dunnett's test analysis, p.ltoreq.0.05; * P is less than or equal to 0.01.

FIG. 20 shows a comparison of ear positions of control plants and transgenic inbred plants overexpressing the invention for each year. By Dunnett's test analysis, p.ltoreq.0.05; * P is less than or equal to 0.01.

FIG. 21 shows a comparison of plant heights for control plants and over-expressed transgenic hybrid plants of the invention for each year. By Dunnett's test analysis, p.ltoreq.0.05; * P is less than or equal to 0.01.

FIG. 22 shows a comparison of ear positions of control plants and over-expressed transgenic hybrid plants of the invention for each year. By Dunnett's test analysis, p.ltoreq.0.05; * P is less than or equal to 0.01.

Detailed Description

The following definitions and illustrations are provided to better define the invention and to guide those of ordinary skill in the art in practicing the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless specifically stated otherwise, the techniques employed and contemplated herein are standard methods well known to those skilled in the art to which the present invention pertains. The materials, methods, and examples are illustrative only and not intended to limit the scope of the present invention in any way.

As used herein, "plant" includes whole plants, transgenic plants, meristems, parts of plants, plant cells, and progeny thereof. Parts of a plant include, but are not limited to, leaves, stems, tubers, roots, flowers (including, for example, bracts, sepals, petals, stamens, carpels, anthers, ovules, etc.), fruits, embryos, endosperm, seeds, pollen, meristematic tissue, callus, protoplasts, microspores, and the like.

As described herein, useful plant species generally encompass higher plant species suitable for transgenic technology, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, scouring rush, gymnosperms, pinus, bryophytes, and multicellular algae.

In some embodiments, the parts or plant cells of the plants of the invention are regenerable. In other embodiments, the parts or plant cells of the plants of the invention are non-regenerable.

In a specific embodiment, the plants suitable for the present invention are selected from the following: corn, sorghum, soybean, wheat, rice, cotton, canola, cabbage, canola, mustard, barley, rye, oat, millet, tomato, sunflower, potato, peanut, sweet potato, cassava, sugar beet, sugarcane, tobacco, arabidopsis, and the like. Preferably, the plant suitable for use in the present invention is maize or rice.

As used herein, the term "transgene" refers to a polynucleotide molecule that is artificially introduced into the genome of a host cell. Such transgenes may be heterologous to the host cell. The term "transgenic plant" refers to a plant comprising the heterologous polynucleotide described above, and transgenic plants include plants regenerated from initially transformed plant cells and progeny transgenic plants from subsequent generation or crossing of the transgenic plant.

Any method known to those skilled in the art can be used to obtain transgenic plants, for example by electroporation or microinjection of plant cell protoplasts into heterologous nucleic acid sequences; or introducing the heterologous nucleic acid sequence directly into the plant tissue cells by DNA microprojectile bombardment (DNA PARTICLE template); or introducing the heterologous nucleic acid sequence into a plant cell using an Agrobacterium tumefaciens (Agrobacterium tumefaciens) host cell.

As used herein, "control plants" refers to plants that do not contain recombinant DNA that confers enhanced traits. Control plants are used to identify and select transgenic plants with enhanced traits. Suitable control plants may be non-transgenic plants of the parental line used to generate the transgenic plants, e.g., wild type plants (WTs). Suitable control plants may also be transgenic plants containing recombinant DNA conferring other traits, e.g., transgenic plants with enhanced herbicide tolerance.

As used herein, "trait" refers to a physiological, morphological, biochemical or physical characteristic of a plant or a particular plant material or cell. In some cases, the characteristic is visible to the human eye, such as seed or plant size, or may be measured by biochemical techniques (e.g., detecting protein, starch, certain metabolites, or oil content of seeds or leaves) or by observing metabolic or physiological processes (e.g., by measuring tolerance to water deprivation or specific salt or sugar concentrations) or by measuring the expression level of one or more genes (e.g., using Northern analysis, RT-PCR, microarray gene expression assays, or reporter gene expression systems) or by agricultural observations (e.g., tolerance to hyperosmotic stress and yield). Any technique can be used to measure the amount, comparison level or difference of any selected chemical compound or macromolecule in the transgenic plant.

As used herein, "gene" or "gene sequence" refers to the partial or complete coding sequence of a gene, its complement, and its 5 'and/or 3' untranslated regions. Genes are also genetic functional units and are physically specific segments or sequences of nucleotides of molecules along the DNA (or RNA, in the case of RNA viruses) that are involved in the production of polypeptide chains. The latter may undergo subsequent processing, such as chemical modification or folding, to obtain a functional protein or polypeptide. By way of example, a transcriptional regulatory gene encodes a transcriptional regulatory polypeptide, which may be functional or require processing to act as a trigger for transcription.

As used herein, the term "nucleic acid" refers to any polymer comprising deoxyribonucleotides or ribonucleotides, including but not limited to modified or unmodified DNA, RNA, the length of which is not subject to any particular limitation. For nucleic acids used to construct the recombinant construct, DNA is preferred, which is more stable and easier to manipulate than RNA.

The term "DNA" refers to a double-stranded DNA molecule of genomic or synthetic origin, i.e., a polymer of deoxyribonucleotide bases or a nucleotide molecule, read from the 5 'end (upstream) to the 3' end (downstream). The term "nucleotide sequence" refers to the sequence of nucleotides of a DNA or RNA molecule that is typically displayed from the 5 '(upstream) end to the 3' (downstream) end.

Methods known to those of skill in the art may be used to isolate and describe the DNA molecules or fragments thereof. For example, PCR (polymerase chain reaction) techniques can be used to amplify specific starting DNA molecules and/or to generate variants of the original molecule. The DNA molecule or fragment thereof may also be obtained by other techniques, such as direct synthesis of the fragment by chemical means (e.g. an automated oligonucleotide synthesizer).

The term "transcript" refers to a product of transcription of a DNA nucleotide sequence catalyzed by an RNA polymerase. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as a primary transcript, or it may be RNA obtained by post-transcriptional processing of a primary transcript, referred to as mature RNA.

The term "isolated" means that the molecule is at least partially separated from other molecules to which it is normally attached in its native or natural state.

In some embodiments, the term "isolated nucleic acid molecule" refers to a nucleic acid molecule that is at least partially separated from nucleic acids that normally flank the nucleic acid molecule in an autologous or natural state. Thus, nucleic acid molecules fused to regulatory or coding sequences to which they are not normally linked are considered isolated herein, as a result of recombinant techniques. Such molecules are considered isolated even when integrated into the chromosome of a host cell or present in a nucleic acid solution with other nucleic acid molecules.

As used herein, a "polypeptide" comprises a plurality of consecutive polymeric amino acid residues, e.g., at least about 15 consecutive polymeric amino acid residues. Typically, a polypeptide comprises a series of polymeric amino acid residues that are transcriptional modulators or domains or portions or fragments thereof. Furthermore, the polypeptide may comprise: (i) a localization domain; (ii) an activation domain; (iii) an inhibitory domain; (iv) an oligomerization domain; (v) a protein-protein interaction domain; (vi) a DNA binding domain; or other portion. The polypeptide optionally comprises modified amino acid residues, naturally occurring amino acid residues not encoded by a codon, non-naturally occurring amino acid residues.

As used herein, "protein" refers to a series of amino acids, oligopeptides, peptides, polypeptides, or portions thereof, whether naturally occurring or synthetic.

The term "isolated polypeptide" refers to a polypeptide, whether naturally occurring or recombinant, that is present in a cell (or extracellular) in a greater amount than the polypeptide in its natural state in a wild-type cell, e.g., in an amount of greater than about 5% or greater than about 10% or greater than about 20% or greater than about 50% or greater, alternatively expressed as: the content is 105%,110%,120%,150% or more relative to the wild-type polypeptide homogenized at 100%. This is not the result of the natural response of wild type plants. In addition, the isolated polypeptides are separated from other normally associated cellular components, for example, using various protein purification methods.

As used herein, "percent identity", "% identity" or "percent identity" is a comparison between amino acid sequences or nucleotide sequences, as determined by comparing two sequences optimally aligned over a window of comparison (e.g., for optimal alignment with another nucleotide sequence or amino acid sequence, gaps can be introduced in the first nucleotide sequence or amino acid sequence). Those skilled in the art know how to calculate the percent identity between two sequences, for example, by various software such as Clustal, bestfit, blast, fasta. Percent identity is determined by: the number of positions of the same nucleotide or amino acid in both sequences is calculated, divided by the full length of the reference sequence (excluding gaps introduced into the reference sequence by the alignment process), and multiplied by 100%. The reference sequence may be as shown in SEQ ID NO.1, SEQ ID NO. 3 or SEQ ID NO.5, or SEQ ID NO. 2, SEQ ID NO. 4 or SEQ ID NO. 6. One of the other sequences may have one or more amino acid/nucleotide insertions, substitutions and deletions relative to the reference sequence.

In some embodiments, the nucleotide sequence of the present invention has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the nucleotide sequence set forth in SEQ ID NO. 1.

In some embodiments, the polypeptide sequences of the invention have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 2.

As used herein, the term "stringent conditions" or "stringent hybridization conditions" includes conditions under which a probe hybridizes to a target sequence with a higher degree of detectability than other sequences (e.g., at least twice as high as background). Stringent conditions will be different depending on the sequence and circumstances. By adjusting the stringency of hybridization and/or wash adjustments, target sequences up to 100% complementary to the probe can be detected. Alternatively, the stringency can be adjusted so that some mismatches exist in the sequences, thereby detecting target sequences of lower identity. Hybridization specificity depends on the wash step after hybridization, the key factors being the salt concentration and temperature in the wash solution. Both the temperature and salt concentration may be varied, or the temperature or salt concentration may remain unchanged while another variable is changed, as desired. Suitable stringent conditions for promoting DNA hybridization are known to those skilled in the art. Examples of low stringency conditions are: hybridization was performed at 37℃in a buffer containing 30-35% formamide, 1M NaCl, 1% SDS, and washing with 1-2 XSSC (sodium chloride/sodium citrate) at 50℃to 55 ℃. Examples of moderately stringent conditions are: hybridization was performed at 37℃in a buffer containing 40-45% formamide, 1M NaCl, 1% SDS, and washed with 0.5-1 XSSC at 55℃to 60 ℃. Examples of highly stringent conditions are: hybridization was performed at 37℃in a buffer containing 50% formamide, 1M NaCl, 1% SDS, and washed with 0.1 XSSC at 60℃to 65 ℃. For a detailed description of nucleic acid hybridization see Haymes et al, nucleic Acid Hybridization, APRACTICAL APPROACH, IRL Press, washington, DC (1985); and Sambrook et al Current Protocols in Molecular Biology, john Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.

As used herein, "insertions," "deletions," or "substitutions" of one or more nucleotides or amino acids, where the insertions, deletions, and/or substitutions do not impair the function of the original sequence (e.g., in this context, refer to functions that still preserve drought resistance and/or increase crop yield under drought conditions). Those skilled in the art are aware of methods for accomplishing one or more nucleotide/amino acid insertions, deletions, and/or substitutions in the original sequence while preserving the biological function of the original sequence. Such insertions, deletions and/or substitutions are chosen to be made in non-conserved regions; or modifying a nucleotide by "silent variation" without altering the polypeptide encoded by the nucleotide based on the degeneracy of the genetic code; alternatively, one amino acid in a protein is replaced by another amino acid of similar nature by "conservative substitutions" without affecting the biological function of the protein. Conservative substitutions may occur within the following groups: 1) Acidic (negatively charged) amino acids such as aspartic acid and glutamic acid; 2) Basic (positively charged) amino acids such as arginine, histidine and lysine; 3) Neutral polar amino acids such as glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; and 4) neutral nonpolar (hydrophobic) amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Conservative substitutions for amino acids within a native protein or polypeptide may be selected from other members of the group to which the naturally occurring amino acid belongs. For example, the group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine and isoleucine; amino acid groups having aliphatic-hydroxyl side chains are serine and threonine; amino acid groups having amide-containing side chains are asparagine and glutamine; amino acid groups having aromatic side chains are phenylalanine, tyrosine and tryptophan; amino acid groups with basic side chains are lysine, arginine and histidine; and the group of amino acids having sulfur-containing side chains are cysteine and methionine. The natural conserved amino acid substitution groups are: valine-leucine, valine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine valine, aspartic acid-glutamic acid and asparagine-glutamine.

As used herein, the term "recombinant" refers to DNA and/or protein and/or organism forms that are not normally found in nature and are thus produced by human intervention. Such human intervention may result in recombinant DNA molecules and/or recombinant plants.

In some embodiments, the recombinant vector further comprises a promoter, terminator, regulatory sequence, selectable marker, and/or any other sequence necessary for expression thereof in a host cell operably linked to the nucleotide sequence. In some specific embodiments, the recombinant vector is a plasmid.

As used herein, the term "promoter" generally refers to a DNA molecule that is involved in the recognition and binding of RNA polymerase II and other proteins (trans-acting transcription factors) to initiate transcription.

In some embodiments, the promoter may be initially isolated from the 5 'untranslated region (5' utr) of the genomic copy of the gene; alternatively, the promoter may be a synthetically produced or manipulated DNA molecule. In some embodiments, the promoter may also be chimeric, i.e., a promoter produced by fusion of two or more heterologous DNA molecules. Plant promoters include promoter DNA obtained from plants, plant viruses, fungi, and bacteria (e.g., agrobacterium). In some embodiments, the promoter is a developmentally regulated, organelle specific, tissue specific, inducible, constitutive, or cell specific promoter.

As used herein, "operably linked" is used interchangeably with "operably linked" to refer to a functional linkage between a first sequence (e.g., a promoter) and a second sequence (e.g., a gene of interest), wherein the promoter sequence initiates and mediates transcription of the second sequence. Typically, the two sequences operably linked are contiguous. Those skilled in the art know how to select promoters, terminators and other sequences necessary for expression of a gene in a host cell.

In some specific embodiments, expression of a gene is controlled by a so-called "strong" promoter (i.e., a promoter with high transcriptional potential such that the gene is strongly expressed).

As used herein, "host cell" refers to a cell that contains a recombinant vector and supports replication and/or expression of the expression vector. The host cell may be a prokaryotic cell (e.g., an E.coli cell, an Agrobacterium tumefaciens cell) or a eukaryotic cell (e.g., a yeast, insect, plant or animal cell).

In some embodiments, the host cell is preferably a monocot or dicot cell, including, but not limited to, cells from maize, sorghum, soybean, wheat, rice, cotton, canola, cabbage, canola, mustard, barley, rye, oat, millet, tomato, sunflower, potato, peanut, sweet potato, cassava, sugar beet, sugarcane, tobacco, arabidopsis. Preferably, the host cell is a maize cell or a rice cell; more preferably, the host cell is a maize cell.

As used herein, "introducing" a nucleic acid molecule or expression vector into a plant or plant cell refers to transfecting, transforming, transducing or incorporating the nucleic acid molecule or recombinant expression vector into a host cell such that the nucleic acid molecule is capable of autonomous replication or expression in the host cell.

In some embodiments, the introduced nucleic acid molecule is integrated into host cell genomic DNA (e.g., chromosomal, plasmid, plastid, or mitochondrial DNA), and expression of the nucleotide sequence is under the control of a regulatory promoter region. In other embodiments, the introduced nucleic acid molecule is not integrated into the cell genome.

The CRISPR/Cas gene editing systems described herein include all components required for, for example, cas9 or modified Cas9 enzymes, guide RNAs, and/or homology-directed repair templates, and the like; comprising (a) a CRISPR/Cas system nucleotide sequence or a nucleotide sequence encoding a CRISPR/Cas system nucleotide sequence and/or (b) a nucleotide sequence encoding a CRISPR/Cas enzyme, which sequences may be comprised in one or more recombinant viral vectors. Wherein the nucleotide sequence of (a) may be located on the same or a different recombinant viral vector as the nucleotide sequence of (b).

In some embodiments, the viral vector may be a retroviral vector, optionally a lentiviral vector, a baculovirus vector, a herpes simplex viral vector, an adenovirus vector, an adeno-associated virus (AAV) vector (e.g., AAV8 vector), or a poxvirus (e.g., vaccinia virus).

In some embodiments, (a) the CRISPR/Cas system nucleotide sequence or the nucleotide sequence encoding the CRISPR/Cas system nucleotide sequence and/or (b) the nucleotide sequence encoding the CRISPR/Cas enzyme may be delivered to a cell of an organism by a liposome, nanoparticle, exosome, microvesicle, or gene gun.

In some specific embodiments, the preferred CRISPR/Cas enzyme is a type II CRISPR/Cas enzyme, preferably a type II Cas9 enzyme or a biologically active fragment or derivative thereof.

The corn 'early 15' gene sequence related to the application is Zheng 58, and the Zea mays (B73 RefGen_v3) version codes as follows: GRMZM2G171365, zea mays (Zm-B73-REFERENCE-GRAMENE-4.0) version coded as Zm00001d048474, and Zea mays (Zm-B73-REFERENCE-NAM-5.0) version coded as Zm00001eb403750. Because the same segment of DNA sequence of corn can produce different transcripts and translate different proteins, the different transcripts produced by the segment of sequence and the translated different proteins are all within the protection scope of the application.

The invention uses the gene editing method to knock out the early 15 gene of corn, and can obtain late maturing corn plants; and the maize 'early 15' gene is overexpressed in maize, so that maize plants with early maturing and short stalks can be obtained. Therefore, the invention has important theoretical guiding significance and production application value for cultivating corns with early flowering and mature periods and reduced plant height and spike position.

The invention has the following technical effects:

In the embodiment of the invention, the maize plant with early maturing and short stalk can be obtained by over-expressing the maize 'early 15' gene in maize. Compared with the traditional breeding mode, the flowering phase and the mature phase of the transgenic corn with the 'early 15' gene over-expression are both obviously and stably advanced, and the plant height and the ear position are both obviously and stably reduced. Therefore, the invention provides gene resources for cultivating and improving corn, and provides theoretical basis for elucidating important roles of early 15 gene in plant growth and development, especially early maturing and short stalk. In conclusion, the invention widens the sources of the excellent alleles available in plant breeding and provides a new idea for obtaining the excellent alleles; greatly shortens the selection process of the excellent allele and provides possibility for the application of the excellent allele.

Detailed Description

The maize acceptor material inbred used in the following examples was ND101; the agrobacterium strain is EHA105.

Example 1 construction of over-expression vector design and creation of transgenic Material

1.1 Carrier frameworks

PBCXUN: the HYG gene is modified based on a vector pCXUN (NCBI GenBank: FJ 905215) and replaced by Bar through XhoI locus; the Bar gene is from pCAMBIA3301, pCXUN, the promoter for promoting the expression of target gene is corn Ubiquitin-1, and the terminator is T-nos.

The vector map is shown in FIG. 4.

1.2 Construction of the "early 15" Gene overexpression vector

First, RNA was extracted from the V4 leaf of Zheng 58 of maize variety using a magnetic bead method plant total RNA extraction kit (Peking Baitaike Biotechnology Co., ltd., cat. No. AU 3402) according to the manufacturer's instructions, and then the RNA was reverse transcribed into cDNA using High-CAPACITY CDNA REVERSE Transcription Kit (Thermo Scientific Co., ltd., cat. No. 4368814), and a whole genome cDNA library was constructed. Then, the cDNA library was sequenced, a plasmid containing the complete CDS sequence of the "early 15" gene was selected by bioinformatics method with reference to published B73 sequence and gene annotation, and the CDS sequence of the "early 15" gene was obtained by restriction enzyme digestion with the restriction enzyme Quick cut SfiI (TaKaRa, cat# 1637) (GRMZM 2G171365cDNA (Zheng 58)). The purified "early 15" CDS sequence was ligated with pBCXUN vector (NCBI GenBank: FJ905215, see, e.g., plant Physiol.150 (3), 1111-1121 (2009)) to give recombinant expression vector pBCXUN- "early 15" (see FIG. 5). The recombinant expression vector is used to transform colibacillus, positive clone is screened, and the complete early 15 gene is confirmed by sequencing.

1.3 Preparation of transgenic maize overexpressing the "early 15" Gene

Recombinant expression vector pBXCUN- "early 15" is introduced into agrobacterium EHA105 strain to obtain recombinant strain. Then, the recombinant bacteria are led into maize inbred line ND101 by an agrobacterium-mediated method to obtain a T ₀ generation transgenic plant.

And taking seedling leaves of T ₀ generation transgenic plants, and extracting genome DNA. PCR amplification was performed using the genomic DNA as a template, and primer Ubip-F (5 'end of Ubi1P promoter against recombinant expression vector pBXCUN- "early 15") and primer Nos-R (3' end of Nos terminator against recombinant expression vector pBXCUN- "early 15"). Genomic DNA from maize inbred ND101 seedling leaves served as negative control, and plasmid of recombinant expression vector pBXCUN- "early 15" served as positive control.

Ubip-F：5’-TTTTAGCCCTGCCTTCATACGC-3’(SEQ ID NO:4)；

Nos-R：5’-AGACCGGCAACAGGATTCAATC-3’(SEQ ID NO:5)。

The PCR amplification products were detected by agarose gel electrophoresis. The result shows that the transgenic plant and the plasmid can amplify a single band with the size of 1291bp, and the parent ND101 does not amplify a corresponding band, which indicates that the early 15 gene is successfully transferred into the transgenic plant.

Selfing the identified T ₀ generation transgenic plant to obtain a T ₁ generation transgenic plant offspring; selfing the progeny of the transgenic plant of the generation T ₁ to obtain the progeny of the transgenic plant of the generation T ₂; selfing the progeny of the transgenic plant of the generation T ₂ to obtain the progeny of the transgenic plant of the generation T ₃; the positive transgenic plants are identified in each generation by adopting the PCR amplification method, and then selfing is carried out. 3 representative T ₃ -generation homozygous transgenic lines (i.e., early 15-1, early 15-2, early 15-3) were selected for subsequent functional analysis experiments.

1.4 Detection of expression level of early 15 Gene

This example uses 3 representative T3 generation homozygous transgenic lines (i.e., early 15-1, early 15-2, early 15-3) and maize inbred ND101 (WT) as test plants.

First, RNA of V4 leaf of the plant to be tested was extracted using a magnetic bead method plant total RNA extraction kit (Peking Baitaike Biotechnology Co., ltd., cat. No. AU 3402) according to the manufacturer's instructions, and then the RNA was reverse transcribed into cDNA using High-CAPACITY CDNA REVERSE Transcription Kit (Thermo Scientific Co., ltd., cat. No. 4368814).

Then, real-time fluorescent quantitative PCR amplification was performed using SYBR Premix Ex TaqTM II (TLI RNASEH Plus) kit (Takara Co., ltd., cat. No. RR 820A) and specific primers "early 15" -Q-F (5'-CTCCGCCGCCGCCTCGTG-3') (SEQ ID NO: 6) and "early 15" -Q-R (5'-CGTCCATTCCGTCGTCCTTC-3') (SEQ ID NO: 7) using cDNA as a template to detect the expression level of the "early 15" gene. The cDNA of maize inbred ND101 (WT) was used as a control. The maize action gene is used as an internal reference gene, and the detection primers are as follows: zmActin-rF:5'-GAGCTCCGTGTTTCGCCTGA-3' (SEQ ID NO: 8) and ZmActin-rR:5'-CAGTTGTTCGCCCACTAGCG-3' (SEQ ID NO: 9). The reaction procedure for fluorescent quantitative PCR amplification is shown in Table 1 below.

TABLE 1 reaction procedure for fluorescent quantitative PCR amplification

The results of the fluorescent quantitative PCR are shown in FIG. 6. As can be seen from FIG. 6, the expression level of "early 15" in transgenic lines early 15-1, early 15-2 and early 15-3 is significantly higher than that of the maize inbred line ND101 (WT) of the control plant. These results indicate that the "early 15" gene was successfully overexpressed in the T ₃ generation homozygous transgenic lines early 15-1, early 15-2, and early 15-3.

EXAMPLE 2 creation of Gene editing vector and Gene editing Material

2.1 Gene editing vector backbone

The vector pBUE C used in this example was modified based on pBUE to construct an existing final vector pBUE C by replacing the resistance gene SpR with the gene ccdB, which is a lethal gene of E.coli F-plasmid negative strain, at BsaI site. After the vector is transferred into DH5 alpha, negative bacteria cannot grow, and the positive rate can be obviously improved to more than 90%. pBUE A related study result has been published (Xing et al BMC Plant biol.2014nov 29;14 (1): 327.) by professor of the functional genome of crops of Chinese university of agriculture in cooperation with molecular breeding research center, professor laboratory design of biological institute Chen Jijun.

The structure of gene editing vector pXUE C is shown in FIG. 7.

2.2 Design of early 15 Gene target and construction of Gene editing vector

The single-target gene knockout vector is designed and constructed 15M earlier, and a specific single-target sequence GGTGACCTGGCGGCTGGTC is constructed to a vector pBUE C. And (3) introducing the recombinant expression vector into an agrobacterium EHA105 strain to obtain recombinant bacteria. Then, the recombinant bacteria are led into maize inbred line ND101 by an agrobacterium-mediated method to obtain a T ₀ generation transgenic plant.

2.2.1 Target design the website http:// crispor.tefor.net/search candidate targets were designed using the gene editing vector, and then screening was performed on candidate targets. Because the target design website is based on the inbred line which has completed sequencing, and is different from the transgenic receptor material, in order to ensure the gene editing efficiency, the target verification of the transgenic receptor material is required. The wild DNA of the maize inbred line of the receptor material is taken as a template, a specific primer is designed by referring to the DNA sequence of the target gene, sequencing verification is completed on a target sequence section covered by the receptor material, and the target sequence is completely consistent with the receptor material.

2.2.2 Target verification

The acceptor maize inbred line ND101 wild type DNA is used as a template, 30 mu l system PCR amplification is carried out by KOD enzyme, the primer is 15M- (-172) F & 15M-229R, and the PCR system and the Touch Down PCR are as follows. And 5. Mu.l of PCR product is taken for agarose gel electrophoresis detection, the fragment size of the PCR product is correct and specific, the rest products are directly sent and detected, the sequencing primer is 15M- (-172) F earlier, and whether the target sequence is completely matched with the receptor material is verified according to the sequencing result. And if the carrier is completely matched, carrying out the next carrier design construction.

Target verification primer:

Early 15M- (-172) F:5'-CCCCAATTCGGATAACAAACC-3' (SEQ ID NO: 10)

Early 15M-229R:5'-CTCGAACTCGAAGGGAAGACG-3' (SEQ ID NO: 11)

PCR amplification system:

PCR amplification procedure:

PCR amplified sequence (SEQ ID NO: 12):

2.2.3 vector construction

After confirming the target sequence, synthesizing a primer to construct a single target carrier: directly annealing the oligonucleotide chain containing the target sequence by using a PCR instrument and then connecting the oligonucleotide chain to the digested gene editing vector pBUE C; the principle and the construction method refer to articles published by a teacher of China agricultural university Chen Jijun. (Xing et al BMC Plant biol.2014Nov 29;14 (1): 327.).

The single target carrier construction primers were as follows:

CAU0574-BsF：5’-GGCGGGTGACCTGGCGGCTGGTC-3’(SEQ ID NO:13)

CAU0574-BsR：5’-AAACGACCAGCCGCCAGGTCACC-3’(SEQ ID NO:14)

The recombinant expression vector is used for transforming escherichia coli, positive clones are screened, plasmids are extracted and then sent to a sequencing company for sequencing, sequencing results are compared with target targets for analysis, the fact that the vector contains complete target sequences with PAM at the 3' end is ensured, the fact that the correct target sequences are introduced into pBUE C is indicated, and cloning construction is completed.

2.3 Creation of "early 15" Gene editing Material in ND101

The recombinant gene editing vector is introduced into an agrobacterium EHA105 strain at 15M to obtain recombinant bacteria. Then, the recombinant bacteria are led into a maize inbred line ND101 through an agrobacterium-mediated method, and T ₀ generation transgenic seedlings are obtained by screening aiming at Bar resistance, thus obtaining T ₀ generation transgenic plants.

And after genetic transformation and emergence of seedlings, taking seedling leaves of the T ₀ -generation transgenic plants, and extracting genome DNA. Detecting the copy number of the resistance gene Bar by taking the genome DNA as a template, and screening to obtain a transgenic positive plant; and then selecting target spot verification primer early 15M- (-172) F & early 15M-229R to carry out gene editing detection on the target gene sequence, and carrying out PCR amplification. The PCR product is directly sent to a company for sequencing, the sequencing primer is 15M- (-172) F, the mutation type after gene editing is further analyzed, and mutant strains are screened.

Bar gene copy number and gene editing type are detected for their progeny T ₁ generation plants, single plant phenotype screening is performed at T ₁, and strains with phenotypes (early 15M-1, early 15M-2 and early 15M-3) are selected for field testing at T ₂.

Example 3 flowering phase analysis of the over-expressed transgenic maize and Gene editing Material of the invention

3.1 Analysis of the growth period of overexpressed transgenic maize

The materials used in the test of the overexpression transgenic maize inbred line are pBCXUN- "early 15" lines early 15-1, early 15-2 and early 15-3, and ND101 is used as a control plant. The test material used in the over-expression transgenic corn hybrid test is that pBCXUN- "early 15" strain 3T3 generation homozygous transgenic strains are used as male parent and hybridized with T13 to obtain 3 transgenic homozygous strains F1 (namely F1-early 15-1, F1-early 15-2 and F1-early 15-3), and F1-ND101 obtained by hybridization of ND101 and T13 is used as control plant. Many years of multi-point experiments were performed on both inbred lines and hybrids. Early 15-1, early 15-2, early 15-3 and WT (ND 101) were planted in 2015 at 1 test point (long sunlight condition: princess, 2 replicates per point), 2020 at 2 test points (long sunlight condition: princess, short sunlight condition: unsealed summer sowing, 3 replicates per point), 2021 at 2 test points (long sunlight condition: princess, short sunlight condition: unsealed summer sowing, 3 replicates per point), 2022 at 1 test point (long sunlight condition: princess, 3 replicates per point). F1-early 15-1, F1-early 15-2, F1-early 15-3 and WT (F1-ND 101) were planted in 8 points (long sunshine condition: princess, baotou, zhuozhou, anyang spring sowing, yinchuan; short sunshine condition: anyang summer sowing, unsealed summer sowing, 2 repetitions per point), 2 points (long sunshine condition: princess, short sunshine condition: unsealed summer sowing, 1 repetition per point) in 2021, and 1 trial points (long sunshine condition: princess, 2 repetitions per point) in 2022, respectively, in 2015. The field traits of each strain, including silking period, pollen scattering period, maturation period, plant height and ear height, were investigated from seedling stage to maturation stage.

FIG. 8 shows the results of comparison of the number of days of pollen shed for different lines of "early 15" overexpressing transgenic maize inbred line test versus control plants. As shown in fig. 8, the 3 inbred lines in 2015 had an average 6.68 days earlier in the powder dispersion day, the 3 lines in 2020 had an average 5.61 days earlier, and the 3 lines in 2021 had an average 6.42 days earlier than the control plant ND101, all reaching very significant levels. Overall, 3 "early 15" overexpressing transgenic maize inbred lines were 15-1, 15-2 and 15-3 earlier in the number of powder days in 2015, 2020 and 2021 than the control group, and the inbred lines were 6.24 days earlier on average for the three-year trial of powder days, reaching very significant levels.

FIG. 9 shows the results of comparison of the number of days of laying of different lines in the "early 15" overexpressing transgenic maize inbred line test with control plants. As shown in fig. 9, the number of days of drawing of 3 inbred lines in 2015 was 6.78 days earlier on average, 5.35 days earlier in 2020, 6.93 days earlier in 2021, on average, than in the control plant ND101, all reaching very significant levels. Overall, 3 "early 15" overexpressing transgenic maize inbred lines were 15-1, 15-2 and 15-3 all advanced in 2015, 2020 and 2021 days of silk withdrawal over the control group, and the inbred lines were 6.36 days in average in three years of trial silk withdrawal, reaching very significant levels.

FIG. 10 shows the results of comparison of the number of days of maturity of different lines tested with the "early 15" overexpressing transgenic maize inbred line with the control plants. As shown in FIG. 10, 3 "early 15" overexpressed transgenic maize inbred lines were 9.23 days, 8.90 days earlier than the control group by the maturity days of 15-1, 15-2 and 15-3 years earlier than the control group, respectively, with an average 9.0 days earlier than the 3 inbred maturity days, reaching very significant levels.

FIG. 11 shows the results of comparison of the number of days of pollen shed for different lines of "early 15" overexpressing transgenic maize hybrid experiments with control plants. As shown in FIG. 11, the average of the number of days of pollen dispersion for 3 transgenic maize hybrids in 2015 was 7.98 days earlier and the average of the number of strains in 2021 was 6.5 days earlier than the control plants F1-ND101, all reaching very significant levels. In general, 3 'early 15' over-expressed transgenic corn hybrid F1-early 15-1, F1-early 15-2 and F1-early 15-3 are all earlier in powder scattering days in 2015 and 2021 than the control group, and the average powder scattering days of the hybrid two-year test are earlier by 7.24 days, so that the extremely remarkable level is achieved.

FIG. 12 shows the results of comparison of the number of days of laying of different lines in the "early 15" overexpressing transgenic maize hybrid test with control plants. As shown in FIG. 12, the average of 7.30 days earlier in 2015, and 7.00 days earlier in 2021, 3 lines of transgenic maize hybrids, compared to control plants F1-ND101, reached very significant levels. In general, 3 'early 15' over-expressed transgenic corn hybrids F1-early 15-1, F1-early 15-2 and F1-early 15-3 had earlier filament drawing days in 2015 and 2021 than the control group, and the average filament drawing days in two-year test of the hybrid were 7.15 days earlier, reaching a very significant level.

FIG. 13 shows the results of comparison of the number of days of maturity of different lines tested with "early 15" overexpressing transgenic maize hybrids with control plants. As shown in FIG. 13, 3 "early 15" overexpressed transgenic maize hybrids F1-early 15-1, F1-early 15-2 and F1-early 15-3 were 8.23 days, 7.90 days, and 8.0 days earlier on average than the control in the maturity period of 2022, respectively, with very significant levels.

As shown in FIG. 14, the milk line of the "early 15" overexpressing transgenic maize hybrid seed had disappeared as compared to control plants F1-ND101, and the milk line of the hybrid control plant seed had just begun.

The results show that under the conditions of long sunlight and short sunlight, the flowering phase and the maturation phase of the 'early 15' overexpression transgenic corn inbred line and the hybrid are both obviously and stably advanced (see fig. 14, 15 and 16).

3.2 Flowering phase analysis of Gene editing materials

Gene editing materials the materials used in the experiments were early 15M strain early 15M-1, early 15M-2 and early 15M-3, with ND101 as the control plant. Early 15M-1, early 15M-2, early 15M-3 and WT (ND 101) were planted in 2021 at 2 trial points (long sunshine condition: princess, short sunshine condition: kaeseen summer sowing, 1 repetition per point). The field traits of each line were investigated from seedling stage to milk stage.

FIG. 17 shows the results of comparison of the number of days of pollen shed for different lines of "early 15" gene-edited maize inbred line test with control plants. As shown in FIG. 17, 3 gene editing lines were each delayed in the days of powdering at 2021 by 15M-1, 15M-2 and 15M-3, compared to the control group. Specifically, 15M-1 was delayed 0.75 days earlier compared to the control plant ND101, which was different from the control plant ND 101. The 15M-2 and 15M-3 delays by 2.75 days and 4.75 days, respectively, compared to the control plant ND101, all reached very significant levels.

FIG. 18 shows the results of comparison of the number of days of laying of different lines in the "early 15" gene-edited maize inbred line test with control plants. As shown in FIG. 18, 3 gene-editing lines were each delayed in the days of drawing of 2021 by 15M-1, 15M-2 and 15M-3, compared to the control group. Specifically, 15M-1 was delayed 1.05 days earlier compared to the control plant ND101, which was different from the control plant ND 101. The 15M-2 and 15M-3 delays 3.55 days and 5.55 days, respectively, compared to the control plant ND101, all reached very significant levels.

The above results indicate that the flowering phase of the "early 15" gene-edited maize inbred is significantly and stably delayed under both long and short day conditions (see fig. 17, 18).

Example 4 plant height and ear position analysis of overexpressing transgenic corn of the invention

FIG. 19 shows the results of comparison of plant heights of different lines tested with "early 15" overexpressing transgenic maize inbred lines with control plants. As shown in fig. 19, the plant height of 3 inbred lines was reduced by 41.26% on average in 2015, 39.17% on average in 2020, and 34.81% on average in 2021, all at very significant levels, compared to the control plant ND 101. Overall, 3 "early 15" overexpressing transgenic maize inbred lines were each reduced in plant height by 15-1, 15-2 and 15-3 in 2015, 2020 and 2021 compared to the control group, and the three year test plant heights of the inbred lines were reduced by 38.41% on average, reaching very significant levels.

FIG. 20 shows the results of comparison of ear position of different lines with control plants in an "early 15" overexpressing transgenic maize inbred line test. As shown in fig. 20, the spike level was reduced by 69.74% on average in the 2015 3 inbred lines, 55.55% on average in the 2020 3 lines, 49.43% on average in the 2021 3 lines, all at very significant levels, compared to the control plant ND 101. Overall, 3 "early 15" overexpressing transgenic maize inbred lines were reduced in spike levels at 2015, 2020, and 2021 by 15-1, 15-2, and 3 as compared to the control group, and the inbred lines were reduced by 58.24% on average for three years of test spike levels, reaching very significant levels.

FIG. 21 shows the results of a comparison of plant heights of different lines tested on "early 15" overexpressing transgenic maize hybrids with control plants. As shown in FIG. 21, the average plant height of 3 transgenic maize hybrids was reduced by 23.98% in 2015 and 29.32% in 2021 compared to control plants F1-ND101, all reaching very significant levels. In general, the plant heights of 3 'early 15' over-expressed transgenic corn hybrid F1-early 15-1, F1-early 15-2 and F1-early 15-3 in 2015 and 2021 are reduced compared with the control group, and the average plant height of the hybrid two-year test is reduced by 26.65 percent, so that the extremely remarkable level is achieved.

FIG. 22 shows the results of a comparison of ear position of different lines of "early 15" overexpressing transgenic maize hybrids with control plants. As shown in FIG. 22, the ear position of 3 transgenic maize hybrids was reduced by 41.71% on average in 2015 and 46.79% on average in 2021 by 3 lines, all at very significant levels, compared to control plants F1-ND 101. Overall, the ears of 3 "early 15" over-expressed transgenic corn hybrids F1-early 15-1, F1-early 15-2 and F1-early 15-3 were reduced in 2015 and 2021 compared to the control, and the average ears of the hybrid two-year test were reduced by 44.25%, reaching a very significant level.

The results show that under the conditions of long sunlight and short sunlight, the plant height and the ear position of the 'early 15' overexpression transgenic corn inbred line and the hybrid are obviously and stably reduced (see fig. 15 and 16).

Reference is made to:

[1]Tapia-Lopez R,Garcia-Ponce B,Dubrovsky J G,et al..An AGAMOUS-related MADS-box gene,XAL1(AGL12),regulates root meristem cell proliferation and flowering transition in Arabidopsis[J].Plant Physiol,2008,146:1182-1192.

[2]Yoo S K,Lee J S,Ahn J H.Overexpression of AGAMOUS-LIKE 28(AGL28)promotes flowering by upregulating expression of floral promoters within the autonomous pathway[J].Biochem Bioph Res Comm,2006,348:929-936.

[3]Pelucchi N,Fornara F,Favalli C,et al..Comparative analysis of rice MADS-box genes expressed during flower development[J].Sexual Plant Reproduction,2002,15(3):113-122.

[4]Lu S J,Wei H,Wang Y,et al..Overexpression of atranscription factor OsMADS15 modifies plant architecture and flowering time in rice(Oryza sativa L.)[J].Plant Mole Bio Rep,2012,30(6):1461-1469.

[5]Alter P,Bircheneder S,Zhou L.Z,et al..Floweringtime-regulated genes in maize include the transcription factor ZmMADS1[J].Plant Physiol,2016,172:389-404.

Claims (25)

1. An isolated nucleic acid molecule comprising a sequence selected from the group consisting of:

1) A nucleotide sequence shown in SEQ ID NO. 1;

2) A nucleotide sequence with at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the nucleotide sequence shown in SEQ ID NO. 1, wherein the encoded polypeptide has the function of regulating plant growth and development;

3) A nucleotide sequence which hybridizes under stringent conditions to the sequence shown in SEQ ID No. 1;

4) Expressing the nucleotide sequence of the same or functionally deleted or mutated protein by substituting and/or deleting and/or adding one or more nucleotides in the nucleotide sequence shown in SEQ ID NO. 1;

5) Different transcripts were produced from the nucleotide sequence shown in SEQ ID No. 1.

2. An isolated nucleic acid molecule comprising a sequence selected from the group consisting of:

1) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 2;

2) A nucleotide sequence encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity to the amino acid sequence shown in SEQ ID NO. 2, wherein the encoded polypeptide has the function of regulating plant growth;

3) A nucleotide sequence encoding an amino acid sequence obtained by substitution and/or deletion and/or addition of one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 2, and a polypeptide encoded thereby has a function of regulating plant growth and development.

3. An isolated nucleic acid molecule comprising a sequence selected from the group consisting of:

1) A nucleotide sequence shown in SEQ ID NO. 3;

2) A nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the nucleotide sequence shown in SEQ ID NO. 3, wherein the encoded polypeptide has the function of regulating plant growth and development;

3) A nucleotide sequence which hybridizes under stringent conditions to the sequence shown in SEQ ID No. 3;

4) Expressing the nucleotide sequence of the same or functionally deleted or mutated protein by substituting and/or deleting and/or adding one or more nucleotides in the nucleotide sequence shown in SEQ ID NO. 3;

5) Different transcripts were produced from the nucleotide sequence shown in SEQ ID NO. 3.

4. An isolated polypeptide, characterized in that it is transcribed and/or expressed from the nucleic acid molecule of any one of claims 1 to 3.

5. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of seq id no:

1) An amino acid sequence shown in SEQ ID NO. 2;

2) An amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity to the amino acid sequence shown in SEQ ID NO. 2, said polypeptide having the function of regulating plant growth;

3) The polypeptide has the function of regulating plant growth and development by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 2.

6. A recombinant vector comprising the nucleic acid molecule of any one of claims 1-3.

7. A host cell comprising the nucleic acid molecule of any one of claims 1-3, or comprising the polypeptide of claim 4 or 5, or comprising the recombinant vector of claim 6.

8. A transgenic plant comprising the nucleic acid molecule of any one of claims 1-3, or comprising the recombinant vector of claim 6.

9. Transgenic plant according to claim 8, wherein the transgenic plant is a monocotyledonous or dicotyledonous plant, preferably a crop plant.

10. The transgenic plant of claim 8 or 9, wherein the transgenic plant is selected from one or more of maize, sorghum, soybean, wheat, rice, cotton, canola, cabbage, canola, mustard, barley, rye, oat, millet, tomato, sunflower, potato, peanut, sweet potato, cassava, sugar beet, sugarcane, tobacco, or arabidopsis; arabidopsis thaliana and maize are preferred.

11. Use of an isolated nucleic acid molecule according to any one of claims 1 to 3 or an isolated polypeptide according to claim 4 or 5 or a recombinant vector according to claim 6 for regulating plant growth.

12. Use of an isolated nucleic acid molecule according to any one of claims 1 to 3 or an isolated polypeptide according to claim 4 or 5 or a recombinant vector according to claim 6 for regulating early maturity and stumps in plants.

13. The use according to claim 12, comprising the steps of:

1) Introducing the nucleic acid molecule of any one of claims 1-3, or the recombinant vector of claim 6, into a plant of interest to obtain an overexpressed transgenic plant; and

2) The plants are grown such that the over-expressed transgenic plants have early maturing and dwarf performance compared to control plants.

14. Use of an isolated nucleic acid molecule according to any one of claims 1 to 3 or an isolated polypeptide according to claim 4 or 5 or a recombinant vector according to claim 6 for regulating late maturity in plants.

15. The use according to claim 14, comprising the steps of:

1) Disruption of the "early 15" gene in plants or genes homologous to the "early 15" gene in other plants to obtain transgenic plants; and

2) The plants are grown such that the transgenic plants have late-maturing properties compared to control plants.

16. The use of claim 15, wherein the disruption is achieved by knocking out or knocking down the "early 15" gene.

17. The use of claim 15, wherein the disruption is effected by a genome editing system of CRISPR/Cas, TALEN, ZFN or other gene editing system.

18. Use of an isolated nucleic acid molecule according to any one of claims 1 to 3 or an isolated polypeptide according to claim 4 or 5 or a recombinant vector according to claim 6 for breeding plants with altered traits.

19. Use according to any one of claims 11-18, wherein the plant is a monocotyledonous or dicotyledonous plant, preferably a crop plant.

20. The use according to claim 19, wherein the plant is selected from one or more of maize, sorghum, soybean, wheat, rice, cotton, canola, cabbage, canola, mustard, barley, rye, oats, millet, tomato, sunflower, potato, peanut, sweet potato, cassava, sugar beet, sugar cane, tobacco, or arabidopsis; arabidopsis thaliana and maize are preferred.

21. A method of modulating plant growth comprising:

1) Introducing the isolated nucleic acid molecule of any one of claims 1-3, or the recombinant vector of claim 6, into a plant of interest to obtain a transgenic plant; and

2) Cultivating the plant, wherein the transgenic plant has the properties of early maturing and dwarfing compared to a control plant.

22. A method of modulating plant growth comprising:

1) Disruption of the "early 15" gene in plants or genes homologous to the "early 15" gene in other plants to obtain transgenic plants; and

2) Cultivating the plant, wherein the transgenic plant has late-maturing performance compared to a control plant.

23. A method of producing a transgenic plant comprising the isolated nucleic acid molecule of any one of claims 1-3 introduced, or the recombinant vector of claim 6, or the isolated polypeptide of claim 4 or 5 expressed recombinantly, comprising the steps of:

1) Obtaining seeds of said transgenic plant;

2) Planting the seeds to obtain the transgenic plant with stable inheritable characters.

24. A method of producing a transgenic plant comprising a gene that disrupts the "early 15" gene in the plant or is homologous to the "early 15" gene in other plants, comprising the steps of:

1) Obtaining seeds of said transgenic plant;

2) Planting the seeds to obtain the transgenic plant with stable inheritable characters.

25. The method of any one of claims 21-24, wherein the plant is selected from one or more of corn, sorghum, soybean, wheat, rice, cotton, canola, cabbage, canola, mustard, barley, rye, oats, millet, tomato, sunflower, potato, peanut, sweet potato, tapioca, sugar beet, sugarcane, tobacco, or arabidopsis thaliana; arabidopsis thaliana and maize are preferred.